In this paper, we present a selective and differential medium, termed Vibrio alginolyticus (VAL) agar, developed for the isolation and identification of V. alginolyticus. The presence of bile salts, high salinity and high incubation temperature allows the selective growth of moderately halophilic Vibrio species. Differentiation of bacteria is achieved by identifying species capable of sucrose fermentation, made visible by the pH indicator bromocresol purple. In this study, all of the 26 strains of V. alginolyticus and only three of the 99 strains representing 30 species (including 19 Vibrio species) other than V. alginolyticus were able to grow in the VAL medium. The remaining three strains could be further differentiated from V. alginolyticus according to colour or the diameter of colonies produced on VAL agar plates. Colonies isolated from shellfish rearing water and infected shrimp through the use of VAL agar plates were all positively identified as V. alginolyticus by conventional tests and 16S rDNA sequencing. The testing of specificity and differentiation capability of VAL shows the potential of the agar as a medium for the primary isolation of V. alginolyticus from pathological and environmental samples.